Thyroid hormone is essential for normal development, growth, neural differentiation, and metabolic regulation in mammals, and is required for amphibian metamorphosis. There are two thyroid hormone receptor (TR) genes, TR? and TR?, with different patterns of expression in development and in adult tissues. The relevance of TR isoform specificity is strongly supported by the distinct phenotypes reported in families heterozygous for dominant negative mutations of the TR? gene and TR? gene. The mechanism of TR isoform-specific action, however, is not well understood. Posttranslational modification of nuclear receptors is being increasingly recognized as an important mechanism of gene regulation. Most proteins modified by small ubiquitin-like modifier (SUMO) are transcription factors and nuclear hormone receptors. Sumoylation requires conjugation of SUMO to a lysine within the consensus recognition motif ?-Lys-X-Glu (? is a large hydrophobic amino acid) of a protein, which rapidly and dynamically modifies proteins involved in cellular processes. We determined that posttranslational modification of TR by conjugation of SUMO to TR? at lysines 283 and 389, and TR? at lysines 50, 146 and 443, plays an essential role in triiodothyronine (T3)-induced gene induction and repression as well as TR isoform-specificity. TR?-SUMO conjugation is ligand-independent and utilizes the E3 ligase PIASxb. TR?-SUMO is ligand-dependent and utilizes predominantly the E3 ligase PIAS1. SUMO1 and SUMO3 conjugation to TR are required for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The role of SUMO1 and SUMO3 in T3-induction in transient functional assays is closely matched to the pattern of TR and co-factor binding in regulation of endogenous genes, as determined by Chromatin Immunoprecipitation (ChIP) assays. Co-repressors play an essential role in thyroid hormone action, and TR sumoylation is important for co-repressor recruitment. We have demonstrated that TR sumoylation is important for thyroid hormone-regulated metabolic actions including adipocyte differentiation and cross-talk with important metabolic regulators including LXR and PPAR?. Specific aims that will be pursued include; 1. Utilize in vitro models to determine the role of TR sumoylation and TR?/TR? isoform-specific actions in metabolic regulation. 2. Determine how sumoylation modulates TR-mediated gene regulation including TR interaction with transcription co-factors, metabolic signaling pathways, and recognition and binding to T3-regulated genes. 3. Evaluate the influence of TR? and TR? sumoylation site mutations on thyroid hormone regulation of metabolism in a mouse model. We will assess the impact of introducing TR? (K389Q) and TR? (K443Q) gene sumoylation mutations on the thyroid hormone axis, tissue level thyroid status, and metabolic regulation. Understanding the role of TR sumoylation in metabolic regulation and TR-isoform specific action may lead to the identification of novel thyroid hormone signaling targets relevant to therapies for metabolic diseases, including dyslipidemia and obesity.